To a laboratory reactor, in which heterotrophic biofilms were grown on stainless steel coupons under aerobic conditions, sulfate-reducing bacteria (SRB) were added in order to elucidate whether and how these microorganisms were going to establish themselves in the biofilm. Polymerase chain reaction for the dissimilatory sulfite reductase gene and in situ hybridization with probes directed against 16S ribosomal RNA were used to detect the SRB in the biofilm. Both methods proved to be suitable tools for monitoring the SRB in these experiments, which lasted seven days. In a first series of experiments, in which the SRB were added after a biofilm had already developed, the SRB could be detected only one day after addition. No evidence was found that the SRB penetrated the biofilm and established themselves in the anaerobic niches which were present. In a second series of experiments, in which the SRB were inoculated together with a seed of aerobic heterotrophtc microorganisms, the SRB were present in the biofilm over the whole biofilm depth and for the duration of the experiment. The study suggests that colonization of the steel coupons by the SRB added to the bulk fluid is hampered by the already developed biofilm, even though the heterogeneous biofilm structure and anaerobic zones in the biofilm depth offer the possibility for the SRB to penetrate and establish themselves.